Chapter 2
Chemical Syntheses of Hyaluronic Acid Oligosaccharides 1,2
1
1,
Lijun Huang Xiaowei Lu, and Xuefei Huang * 1
Department of Chemistry, The University of Toledo, 2801 West Bancroft Street, MS 602, Toledo, OH 43606 Current address: Anatrace Inc., 434 West Dussel Drive, Maumee, OH 43537 *Corresponding author:
[email protected] 2
The increasing recognition of important biological functions of hyaluronic acid oligosaccharides (sHA) has led to developments of new methodologies for sHA syntheses. Herein, we provide an overview of chemical approaches for assembling these oligosaccharides. Compared with traditional methods, our iterative one-pot strategy provided a new platform for rapid assembly of sHA without resorting to multiple protective group manipulations, anomeric adjustments or intermediate purifications. We expect that the continual development of chemical synthetic methodologies of sHA will provide a facile access to structurally well defined sHA derivatives that will greatly facilitate structure-activity relationship studies.
© 2008 American Chemical Society
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30
Introduction Hyaluronic acid (HA), first discovered by Meyer and Palmer in 1934 (7), belongs to the glycosaminoglycan (GAG) superfamily. As the only unsulfated GAG, HA contains linear tandem disaccharide repeating units of D-glucuronic acid and 2-deoxy-2-7V-acetyl-D-glucose [p-D-Glc/?A-( 1 -»3)-P-D-Glc/?NAc(1—>4)-]. HA is broadly distributed in the extracellular space of vertebrates with the highest concentrations in soft connective tissues (2,3). As a high molecular weight polyanion, an essential role of HA is to participate in a hydrated network with collagen fibers, where it acts as an organizing core to form complex intercellular aggregates. Due to its involvement in a variety of cellular events such as cell adhesion, cell migration, atherosclerosis and wound healing as well as its relatively low toxicity and immunogenicity (4-77), HA has been widely used in clinical applications including in eye surgery and as an intra-articular matrix supplement (6,12).
3
HA can be transformed to hyaluronic acid oligosaccharides (sHA) (~ 2 χ 10 D) in vivo. Biological studies indicated that sHA can have completely distinct novel properties from high molecular weight HA polymers, including stimulation of endothelial cell proliferation and migration (75), stimulation of angiogenesis (7), potentiation of the innate immune system (14-16), inhibition of tumor growth (17) and regulation of multi-drug resistance in cancer cells (18). Several cellular receptors for sHA have been identified. For example, Toll like receptor 4, which is a membrane bound receptor involved in initiation of innate immunity and prevention of early spread of pathogens, has been shown to be activated by sHA tetrasaccharides and hexasaccharides, rather than by the high molecular weight HA (14,15). CD44, a membrane glycoprotein, is involved in many biological events such as cell migration during morphogenesis, angiogenesis, and tumor invasion and metastasis (19-21). HA is the primary ligand for CD44, with the minimum length of a sHA hexasaccharide required for binding (22). The binding affinity between sHA and CD44 increases with longer sHA (23,24). The biological activities of sHA can be sequence specific, an example of which is the report that sHA tetrasaccharides up-regulate the expression of heatshock protein 72 as well as the corresponding mRNA and suppress cell death under stress condition, while the corresponding di-, hexa- and octa-saccharides are inactive (25). To date, the majority of biological studies is performed using
31 a mixture of sHA with various lengths and structures derived from enzymatic degradation of HA isolated from natural sources (26,27). The possibility of highly active contaminants notwithstanding, it is difficult to firmly establish structure-activity relationship (SAR) with a mixture of sHA structures. Access to synthetic sHA with distinct length and sequence can greatly facilitate a systematic investigation of its SAR, providing exciting opportunities for the development of novel therapeutics. sHA can be assembled by either chemoenzymatic or chemical synthesis. Early chemoenzymatic approaches utilized biosynthetic pathways through HA synthases and associated accessory enzymes (28) or transglycosylation reactions catalyzed by hyaluronidases (29). Recently, oxazolidine containing building blocks, which are transition state analogs for hyaluronidases, have been polymerized to yield HA (30-32). In order to control the length of HA from enzymatic reactions, a HA synthase was converted by mutagenesis into two single-action glycosyltransferases (glucuronic acid transferase and Nacetylglucosamine transferase) (33). The alternating stepwise usage of these two novel enzymes led to the construction of a series of monodispersed synthetic sHA. Despite these successes, the inherent substrate specificities of enzymes limit the structural diversity of sHA analogs that can be generated. Chemical synthesis can complement the chemoenzymatic approaches to create greater varities of sHA structures, facilitating SAR studies. In this review, we will focus on the chemical synthesis of sHA and recent progress on iterative one-pot sHA synthesis.
Conventional Syntheses of sHA There are three central factors need to be taken into consideration in designing a synthetic route for sHA: 1) stereocontrolled assembly of the oligosaccharide backbone; 2) introduction of glucuronic acid; and 3) installation of acetamido groups. Two general strategies for sHA assembly are typically adapted depending upon the order of transformations (Scheme 1) (34,35). In the first method, the more reactive glucose is used as a building block with post glycosylation conversion to glucuronic acid via oxidation (Scheme la) (36-42% while the second method utilizes glucuronic acid directly as the glycosyl donor (Scheme lb) (43). Currently, both approaches have been successfiilly applied in preparation of sHA. For the first approach, a selectively removable protective group must be installed on the 6-hydroxyl group of glucose to allow for downstream oxidation state adjustment. As an example, Vliegenthart and coworkers have reported the chemical synthesis of sHA tetrasaccharide 1 with glucuronic acid at the reducing end (Scheme 2) (59). The desired 1,2-frwu linkages were controlled by the presence of participating neighboring groups, i.e., N-phthaloyl (Phth) on
b)
R'O
OR,
Y +
(
2
C
•
OR3 OR3
R4O S
3
R O HO
Oxidation Deprotection
Deprotection
Glycosylation
Aglycon adjustment
R O
»
Glycosylation
—
Aglycon adjustment
2
R,0 R 0
c
a)
Scheme 1.
NRs
NRg
H
OR
\
/
2
H0 C
H
^^0^>-^
nucleophilic substitution
PGO
PGO
Λ
Λ
~° D ^ S ^ O ^ S ^ S T o l
pre-activation
Promoter
PGO
HO^S^SToI
45 yielding glycosylation, presumably due to solubility enhancement of the activated donor at low temperature in the reaction solvent diethyl ether. The Nprotective group of glucosamines did not significantly influence the glycosylation and N-Phth was selected for its high \ 2-trans directing property. 9
Me0 C
PMBO-x
ΡΜΒΟ-Λ
2
A
s
1
M
SSS3S^ ™ « Κ Ε ^ ™ OAc
K3^.° *
OBz
46
OBn
47
NPhth 49: Rj = STol, R = TBS 50: Ri = STol, R = Η 51:R,=OMe,R = H 2
48
N ^ N ]_
T
T
B
P
Bu
2
2
Donor 47 was pre-activated by the promoter /?-TolSOTf, formed in situ through the reaction of AgOTf with p-TolSCl (Scheme 7a). Subsequent addition of acceptor 50 and a sterically hindered non-nucleophilic base 2,4,6-tri'butyl pyrimidine (TTBP) to the reaction formed disaccharide 52, which was deprotected to give disaccharide 53. Disaccharide acceptor 55 with a methoxy group at the reducing end was prepared by reacting 47 with acceptor 51, followed by removal of TBS (Scheme 7b). With all necessary building blocks in hand, one pot syntheses were performed. Pre-activation of donor 47 by p-TolSOTf was followed by addition of acceptor 50 and TTBP. Upon completion of the reaction, addition of acceptor 55, TTBP and promoter p-TolSOTf to the same reaction flask produced sHA tetrasaccharide core 56 in excellent overall yield in just three hours (Table 1, entry 1), which was the only compound that needed purification in this three component one-pot synthesis. sHA core sequences containing an odd number of monosaccharide units can also be synthesized. Two pentasaccharide 57 and 58 with different sequences were constructed through three and four component one-pot reactions in excellent yields (Table 1, entries 2, 3). A hexasaccharide 59 was easily accessed as well in high yield following the pre-activation protocol (Table 1, entry 4). With a near stoichiometric amount of building blocks used for each glycosylation and no oligosaccharide intermediate purification involved, these syntheses can be easily scaled up. Gram quantity of sHA hexasaccharide 59 was obtained within just a few hours in similar yield as the smaller scale reaction. The scalability coupled with the speed of glyco-assembly and high overall yields
46
Scheme 7. a) p-TolSCl, then 50, TTBP 47 + AgOTf
PMBO RO BnO •
b) 47 + 51
AgOTf.TTBP.p-TolSCI
PMBO RO BnO
Ph^O 0
8
Ο
STol
NPhth OBz 52:R = TBS \ HF pyridine 53: R = Η ' Ph^TO
° 8 OBz 54: R = TBS J 55: R = Η
Ο
OMe
NPhth HF pyridine
highlights the advantages of using the iterative one-pot approach for complex oligosaccharide synthesis. After the establishment of sHA core, the next stage of our work was focused on the deprotections and oxidation state adjustments. Careful planning of the deprotection order for various functional groups and strategy for the oxidation of C-6 OH is critical for the success of sHA syntheses. Our initial efforts of removing the three PMB groupsfromhexasaccharides 60 and 63 failed using either CAN or 2,3-dichloro-5,6-dicyano-l,4-benzoquinone (DDQ), although similar reactions have been reported such as CAN oxidation of sHA trisaccharide 28 (Scheme 4) (57). After repeated trials, we found that the presence of TBS moiety was beneficial for deprotection of PMB, as CAN oxidation of hexasaccharide 59 produced triol 61 in good yield (Scheme 8). Conversion of 61 into tricarboxylic acids turned out to be challenging. Several methods such as TEMPO/NaOCl, NaOCI/NaC10 catalyzed by TEMPO, TEMPO/iodobenzenediacetate, and Dess-Martin oxidation followed by NaC10 did not give the desired product presumably due to the need to oxidize multiple hydroxyl groups in the same molecule. Instead, multiple partially oxidized products were often obtained from these reactions. Finally, we discovered that a convenient two step one-pot protocol using TEMPO/NaOCl followed by treatment of NaC10 afforded the desired tri-carboxylic acid, which was isolated as benzyl ester 62 by subsequent treatment with phenyl diazomethane in high yield and good purity (Scheme 8) (48). This new protocol was also found to be compatible with a variety of sensitive functional groups, such as allyl, thioacetal, PMB and isopropylidene. Final deprotections of sHA 62 was carried out by the reaction 2
2
2
Protocol Β A2 (0.81)
50 50
47 47
Β
Β
4
48 55
53 53
59
58
54 - 60%
55%
65%
57 55
3
53
49
A
2
64 - 75%
56
55
50
47
A
1
Yield
Acceptor 3 (A3)
Product
Acceptor 2 (A2)
Acceptor 1 (Al)
Protocol
Entry Donor
Reagents and conditions: a) AgOTf.p-TolSCl, - 65 °C, 10 min; then acceptor, TTBP, 90 min to 0 °C; 15 min, 0 °C; b) acceptor, TTBP, AgOTf, p-TolSCl, - 65 °C - 0 °C in 90 min.
Protocol A
Table 1.
48
sequence of desilylation, hydrogénation, saponification and N-acetylation to produce the sHA hexasaccharide 64. This procedure for deprotection and oxidation state adjustment has also been extended to generate sHA tetrasaccharide 65 and pentasaccharides 66 and 67.
Conclusion It is evident that sHA play important roles in many interesting biological events. Compared with intensive SAR studies on heparin, the importance of sHA is mostly underappreciated in the chemistry community. The developments of new methodologies for the assemblies of sHA would accelerate the exploration of its biological functions. In contrast to traditional syntheses of sHA, the advantages of iterative one-pot synthesis of sHA are obvious with its speed of glycoassembly and scalability. Another important feature of our strategy was the usage of thioglycosides as the glycosyl donors, which are one of the most stable glycosyl donors (49). Furthermore, the development of a two-step one-pot oxidation method via the combination TEMPO/NaOCl and NaC10 has been demonstrated to facilitate the oxidations of multiple hydroxyl groups, which will also benefit synthesis of other GAGs. With chemical approaches, various positions of sHA can be differentiated via protective groups, which can allow for access to structurally varied, well defined sHA derivatives facilitating SAR studies. Nevertheless, sHA synthesis is yet to become routine practice. It is still non-trivial to assemble sHA longer than an octasaccharide unit, which is necessary to study the interaction of sHA with CD44. With the increase in sHA chain length, the solubility and reactivity of the oligosaccharide decrease, which 2
59
74%
CAN
1
Scheme 8. 61
3
2
4
2
TEMPO, NaOCl, NaBr, Bu NBr, NaHC0 , then NaC10 ; PhCHN 62 2
3
2
2
4 4 %from61
2
1) HFpyridine 2) H , Pd(OH) 3) C H N H then A c 0 , MeOH 64
51 can affect the glycosylation as well as deprotection. Further studies are necessary for the continual development of sHA glyco-assembly strategies as well as protecting group chemistry.
References 1. 2. 3. 4.
5.
6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
16. 17. 18. 19.
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